High rates of apoptosis visualized in the symbiont-bearing gills of deep-sea Bathymodiolus mussels Bérénice Piquet, Bruce Shillito, François Lallier, Sébastien Duperron, Ann Andersen To cite this version: Bérénice Piquet, Bruce Shillito, François Lallier, Sébastien Duperron, Ann Andersen. High rates of apoptosis visualized in the symbiont-bearing gills of deep-sea Bathymodiolus mussels. PLoS ONE, Public Library of Science, 2019, 14 (2), pp.e0211499. 10.1371/journal.pone.0211499. hal-02042415 HAL Id: hal-02042415 https://hal.sorbonne-universite.fr/hal-02042415 Submitted on 20 Feb 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. RESEARCH ARTICLE High rates of apoptosis visualized in the symbiont-bearing gills of deep-sea Bathymodiolus mussels 1,2 2 1 2,3,4 BeÂreÂnice Piquet , Bruce Shillito , FrancËois H. LallierID , SeÂbastien Duperron *, Ann C. Andersen1* 1 Sorbonne UniversiteÂ, CNRS, Lab. Adaptation et Diversite en Milieu Marin, AD2M, Team: Adaptation et Biologie des InverteÂbreÂs marins en Conditions Extrêmes (UMR 7144), ABICE, Station Biologique de Roscoff, SBR, Roscoff, France, 2 Sorbonne UniversiteÂ, MNHN, CNRS, IRD, UCN, UA, Lab. Biologie des Organismes a1111111111 et Ecosystèmes Aquatiques BOREA (UMR 7208), Team: Adaptation aux Milieux Extrêmes, AMEX, 7 Quai a1111111111 Saint-Bernard, Paris, France, 3 MuseÂum National d'Histoire Naturelle, CNRS, Lab. MeÂcanismes de a1111111111 Communication et Adaptation des Micro-organismes (UMR 7245), Team: CyanobacteÂries, Cyanotoxines et a1111111111 Environnement, CCE, 12 rue Buffon, Paris, France, 4 Institut Universitaire de France, Paris, France a1111111111 * [email protected] (ACA); [email protected] (SD) Abstract OPEN ACCESS Symbiosis between Bathymodiolus and Gammaproteobacteria allows these deep-sea mus- Citation: Piquet B, Shillito B, Lallier FH, Duperron S, Andersen AC (2019) High rates of apoptosis sels to live in toxic environments such as hydrothermal vents and cold seeps. The quantity visualized in the symbiont-bearing gills of deep-sea of endosymbionts within the gill-bacteriocytes appears to vary according to the hosts envi- Bathymodiolus mussels. PLoS ONE 14(2): ronment; however, the mechanisms of endosymbiont population size regulation remain e0211499. https://doi.org/10.1371/journal. pone.0211499 obscure. We investigated the possibility of a control of endosymbiont density by apoptosis, a programmed cell death, in three mussel species. Fluorometric TUNEL and active Cas- Editor: Clara F. Rodrigues, Universidade de Aveiro, PORTUGAL pase-3-targeting antibodies were used to visualize and quantify apoptotic cells in mussel gills. To control for potential artefacts due to depressurization upon specimen recovery from Received: September 21, 2018 the deep-sea, the apoptotic rates between mussels recovered unpressurised, versus mus- Accepted: January 15, 2019 sels recovered in a pressure-maintaining device, were compared in two species from hydro- Published: February 4, 2019 thermal vents on the Mid-Atlantic Ridge: Bathymodiolus azoricus and B. puteoserpentis. Copyright: © 2019 Piquet et al. This is an open Results show that pressurized recovery had no significant effect on the apoptotic rate in the access article distributed under the terms of the gill filaments. Apoptotic levels were highest in the ciliated zone and in the circulating hemo- Creative Commons Attribution License, which cytes, compared to the bacteriocyte zone. Apoptotic gill-cells in B. aff. boomerang from cold permits unrestricted use, distribution, and reproduction in any medium, provided the original seeps off the Gulf of Guinea show similar distribution patterns. Deep-sea symbiotic mussels author and source are credited. have much higher rates of apoptosis in their gills than the coastal mussel Mytilus edulis, Data Availability Statement: All relevant data are which lacks chemolithoautotrophic symbionts. We discuss how apoptosis might be one of within the manuscript and its Supporting the mechanisms that contribute to the adaptation of deep-sea mussels to toxic environ- Information files. ments and/or to symbiosis. Funding: This research was supported by Institut Universitaire de France project ACSYMB, Universite Pierre et Marie Curie and ARED ReÂgion Bretagne project FlexSyBi (contract number 9127, grant to BP), BALIST program (ANR-08-BLAN-0252), EU projects EXOCET/D (FP6-GOCE-CT-2003-505342), MIDAS (FP7/2007-2013, grant agreement nÊ 603418) and CNRS and IFREMER for cruise PLOS ONE | https://doi.org/10.1371/journal.pone.0211499 February 4, 2019 1 / 21 Apoptosis in gills of hydrothermal vent mussels funding. The funders had no role in study design, Introduction data collection and analysis, decision to publish, or preparation of the manuscript. Symbiosis is of major significance to life on Earth. Because symbiosis between two (or several) partners may straddle the line between cooperation and conflict, each partner theoretically ini- Competing interests: The authors have declared tiates and carries on a continued dialogue with the other partners, to keep some degree of con- that no competing interests exist. trol over the interaction. Many symbioses involve partners from different domains of life, such as a eukaryotic host and a bacterial symbiont. The question then arises: how can reciprocal control occur between such distantly related organisms, and are some of the mechanisms involved universal? One of the mechanisms by which an animal host can control symbiont populations is apoptosis. Apoptosis is a programmed cell death involving three main steps: (1) nuclear condensation and fragmentation, (2) cell-wall budding into apoptotic bodies, and (3) their release and possible phagocytosis by neighboring cells [1,2]. Apoptosis plays multiple roles in normal cell turnover, during development, and in the immune system [3]. Its role in symbiosis has also been documented in the cereal weevil Sitophilus for example, apoptosis plays a role in regulating the densities of the endosymbiotic bacterium Sodalis pier- antonius. The symbiont provides essential amino acids that allow the host to rapidly build its protective exoskeleton. Endosymbionts multiply in young adults, but once the cuticle is built, the symbionts are rapidly eliminated through apoptosis of the host cells that contain them [4]. In corals, bleaching occurs in response to heat stress, when the host releases dinoflagellate sym- bionts through a balance of apoptosis and autophagy: when apoptosis is inhibited, autophagy is initiated as a back-up mechanism, and vice-versa [5]. In addition, apoptosis might act as a post-phagocytic winnowing mechanism in the symbiotic system [6]. At deep-sea hydrothermal vents and cold seeps, the animals that dominate in terms of bio- mass live in association with chemosynthetic bacteria, which sustain most of their nutrition [7±10]. However, the role apoptosis could play in regulating such symbioses has barely been explored. In the vestimentiferan tubeworms Riftia pachyptila, living at vents, and Lamellibra- chia luymesi from cold seeps, the sulfur-oxidizing symbionts are located within cells of the tro- phosome. These cells differentiate and proliferate from the trophosome lobule center, then migrate towards the periphery of the lobule where they undergo apoptosis [11]. Ultrastructural observations of the periphery of trophosome lobules show that the symbionts are digested in vacuoles leading to extensive myelin bodies, after which remnants of symbionts disappear while apoptotic nuclei with clumped chromatin patches appear [11]. Thus, in Riftia as in the weevil, apoptosis appears to be involved in the process of symbiont regulation and could help the host recover the metabolic investment from the symbiotic phase. Deep-sea mussels house very dense populations of endosymbionts inside specialized gill epithelial cells, the bacteriocytes [12,13]. In fact, Bathymodiolus may constitute by far the dens- est microbial habitats, in both vents and seeps, although they usually host a very limited diver- sity of symbiont lineages [7,8]. The relevance to the topic of symbiont control lies in the fact that the association is particularly flexible, with abundances of their symbionts (sulfur- and/or methane-oxidizers) that can change within hours depending on the availability of symbiont substrates in the surrounding water [7,8,14±18]. The symbionts also disappear rapidly if their substrates are absent [17±19]. Ultrastructural studies of the Bathymodiolus gill cells have pointed to intracellular digestion of the symbionts within lysosomes as an important carbon transfer mechanism [20±22] suggesting that the host can access the energy stored in its symbi- onts by killing and digesting them (i.e. a process compared to ªfarmingº). Enzymatic studies involving the detection of acid phosphatases have concluded that some energy from the symbi- onts can also be transferred to the host through molecules leaking from live symbionts (i.e. a process compared to ªmilkingº) [23]. Recent results from whole-gill tissue transcriptomic analyses